Evolution of the Anther Gland in Early-Branching Papilionoids (ADA Clade, Papilionoideae, Leguminosae)

Papilionoideae is the most diverse subfamily of Leguminosae, especially in terms of floral morphology. The ADA clade shows some exciting floral features among papilionoids, such as anther glands. However, the evolution of the anther glands in such early-branching papilionoids remains unknown. Thus, we compared the occurrence, distribution, morphology, and evolutionary history of the anther glands in species of the ADA clade. Floral buds and/or flowers in 50 species were collected from herbarium specimens and investigated using scanning electron and light microscopy and reconstruction of ancestral character states. The anther apex has a secretory cavity, secretory duct, and phenolic idioblast. The lumen shape of the cavity and duct is closely related to the shape of the anther apex. The oval lumen is located between two thecae, the spherical lumen in the prominent anther apex and the elongated lumen in anthers with a long apex. The occurrence of cavities/ducts in the anther in only two phylogenetically closely related subclades is a unifying character -state. The floral architecture is not correlated with cavity/ducts in the anther but is possibly related to the type of pollinator. Future research needs to combine floral morphology and pollination systems to understand the evolution of floral designs and their diversification.

Among the Leguminosae subfamilies, Papilionoideae is considered the most diverse and ecologically successful [2], with a recent history of diversification during the Cenozoic [3,4]. Its diversity is expressed in floral morphology because the presence of papilionaceous flowers characterizes legumes, which also exhibit flowers with other architectural types see [5][6][7][8][9][10].
The early -branching papilionoids comprise plants with exciting flower morphology. They were included in the ADA clade and comprised about 74 species [11,12] (see Figure 1) distributed into three subclades: Amburaneae (eight genera), Angylocalyceae (four genera), and Dipterygeae (four genera) ( Figure 1) [1,10,12]. Some members such as Dipteryx alata and Pterodon pubescens exhibit an unusual condition in the family, which is the presence of glandular appendages in the anther containing a secretory cavity. The secretory cavity Figure 1. Phylogenetic relationships between the three subclades of the ADA clade modified from [1,10,12]. The Angylocalyceae subclade is a sister group to the Amburaneae and Dipterygeae subclades.
The absence of biological information for most species in the ADA clade is due to the sole use of surface analysis (scanning electron microscopy) in the study of flowers without including anatomical sections providing additional information concerning the internal anatomy and intra-and extracellular contents.
In the current study, we present a detailed morphological and evolutionary investigation into the anther glandular appendages of the ADA clade species. We intended to (i) compare the occurrence, distribution and morphology, of the anther glandular appendages in the species of the ADA clade; (ii) to trace the evolutionary history of the secretory structures of the anther based on the recent phylogenetic hypothesis of the ADA clade ( Figure 1) [1,10,12]; (iii) and to evaluate whether the presence of this condition is homologous in some groups.

Figure 1.
Phylogenetic relationships between the three subclades of the ADA clade modified from [1,10,12]. The Angylocalyceae subclade is a sister group to the Amburaneae and Dipterygeae subclades.
The absence of biological information for most species in the ADA clade is due to the sole use of surface analysis (scanning electron microscopy) in the study of flowers without including anatomical sections providing additional information concerning the internal anatomy and intra-and extracellular contents.
In the current study, we present a detailed morphological and evolutionary investigation into the anther glandular appendages of the ADA clade species. We intended to (i) compare the occurrence, distribution and morphology, of the anther glandular appendages in the species of the ADA clade; (ii) to trace the evolutionary history of the secretory structures of the anther based on the recent phylogenetic hypothesis of the ADA clade ( Figure 1) [1,10,12]; (iii) and to evaluate whether the presence of this condition is homologous in some groups.
The hypothesis we tested was that the presence of a secretory cavity/duct in the anther is widely distributed in the ADA clade, and thus, it was acquired by these taxa and can be considered as a synapomorphy for the group. Table 1 summarizes the results from our morphological analysis of the ADA clade species and other selected species. Additional information was obtained from the literature ( Table 1). Because the presence of glands in the anther of this group is an uncommon condition, we analyzed and compared other characteristics related to the anther (for example, apex shape) and to other floral organs (for example, number per whorl and connation) in order to better understand their function in the flower and their evolutionary history.

Flower Morphology
The Amburaneae subclade species (Figures 2 and 3) have flowers with a one-petalled corolla and free stamens (Amburana and Mildbraediodendron), no corolla and free stamens (Cordyla), a papilionaceous corolla and basally united stamens (Dussia and Petaladenium), a non-papilionaceous corolla (five equal petals) and basally united stamens (Myrocarpus), with a papilionaceous corolla and free stamens (Myrospermum), and a non-papilionaceous corolla (widely oval standard) and 10 free homogeneous stamens (Myroxylon). A prominent anther apex occurs in species of Cordyla, Myrospermum, Myroxylon ( Figure 2F,G,I, and Figure 3G,I,J), and a non-prominent anther apex occurs in species of Amburana, Dussia, Mildbraediodendron, Myrocarpus, and Petaladenium (    The Angylocalyceae subclade species (Figures 4 and 5) have flowers with a papilionaceous corolla and basally united stamens (Angylocalyx), a non-papilionaceous corolla that consists of a large vexillum and reduced abaxial petals (Alexa and Castanospermum), a non-papilionaceous corolla that consists of a slightly wider vexillum and four equal abaxial petals and united stamens (Xanthocercis). A prominent anther apex occurs in the two species of Alexa (Alexa grandiflora and A. superba) and Castanospermum australe ( Figures 4E,I and 5C,E); a non-prominent anther apex occurs in species of Angylocalyx, in Xanthocercis zambesiaca and six species of Alexa (Figures 4A-C showing the anther apex with a secretory duct. Note that the lumen shape of the secretory duct follows the shape of the anther apex, rounded in its apical portion (arrowhead) and elongated in the    The Dipterygeae subclade species (Figures 6 and 7) have flowers with a papilionaceous corolla formed by a vexillum, two wings, and two keels, and a monadelphous androecium (Dipteryx, Pterodon, and Taralea); and a non-papilionaceous corolla that consists of a vexillum, two reduced wings, connate and open keels exposing the free stamens (Monopteryx). A prominent anther apex occurs in seven species of Dipteryx, all species of Pterodon and Monopteryx uaucu (Figures 6A,C,D-F,H,J-M and 7L); a non-prominent anther apex occurs in Dipteryx polyphylla, in all species of Taralea and Monopteryx inpae ( Figures 6G and 7A,C,E,G,I-K).   The additional species (Figure 8), Ateleia glazioveana, A. guaraya, Cyathostegia mathewsii, and Uleanthus erythrinoides, exhibit non-prominent anther apices (Figure 8A,C,G,M). In contrast, Candolleodendron brachystachyum and Swartzia langsdorffii have prominent anther apices ( Figure 8E,K,I).

Anther Glands
The anthers of the ADA clade species have three types of glands: secretory cavity, secretory duct, and phenolic idioblast (Table 1).
A secretory duct occurs embedded in the anther apex and extends distally between thecae of the Myroxylon species (Amburaneae subclade) ( Figure 3K). The lumen shape is elongated ( Figure 3K).
In the remaining species, the anther apex does not show a secretory cavity or a secretory duct (Figures 2B,E Phenolic idioblasts are found in the anther apex of Petaladenium, six Alexa species, two Monopteryx species, and in Swartzia langsdorffii (Figures 3M, 4D,F,L, 7M and 8J,L). These species do not have cavities or ducts.

Distribution of Secretory Cavities in the Anther of the Amburaneae and Dipterygeae Subclades
By tracing the evolutionary history of the character "occurrence of secretory cavity/duct in the anther", it was inferred that the presence of cavity/duct in the anther was acquired in most representatives of the Dipterygeae subclade and some of the Amburaneae subclade ( Figure 9).

Distribution of Secretory Cavities in the Anther of the Amburaneae and Dipterygeae Subclades
By tracing the evolutionary history of the character "occurrence of secretory cavity/duct in the anther", it was inferred that the presence of cavity/duct in the anther was acquired in most representatives of the Dipterygeae subclade and some of the Amburaneae subclade (Figure 9).

Correlations between Character -States
The pairings of the character -state reconstructions related to the occurrence of cavity/duct at the anther apex vs. shape of the anther apex indicate a positive correlation between these two characters ( Figure 10). However, it is noteworthy that in 5 of the 20 species (Myrocarpus emarginatus, M. fastigiatus, M. frondosus, Dipteryx polyphylla, and Taralea

Correlations between Character -States
The pairings of the character -state reconstructions related to the occurrence of cavity/duct at the anther apex vs. shape of the anther apex indicate a positive correlation between these two characters ( Figure 10). However, it is noteworthy that in 5 of the 20 species (Myrocarpus emarginatus, M. fastigiatus, M. frondosus, Dipteryx polyphylla, and Taralea cordata), this correlation was negative that is, even without a prominent anther apex, there was a secretory cavity in the anther.

Discussion
Our study highlights how glandular appendages occur in the anther of the ADA clade and provides an opportunity to clarify their enigmatic evolutionary history within

Discussion
Our study highlights how glandular appendages occur in the anther of the ADA clade and provides an opportunity to clarify their enigmatic evolutionary history within early-branching papilionoids. The glandular appendage in the anther has been previously reported in the Dipteryx alata and Pterodon pubescens species of the Dipterygeae subclade [7,13], and also occurring in the species of the Amburaneae subclade.

Discussion
Our study highlights how glandular appendages occur in the anther of the ADA clade and provides an opportunity to clarify their enigmatic evolutionary history within early-branching papilionoids. The glandular appendage in the anther has been previously reported in the Dipteryx alata and Pterodon pubescens species of the Dipterygeae subclade [7,13], and also occurring in the species of the Amburaneae subclade.

Distribution and Location of Secretory Cavities/Ducts
The large ADA clade comprises morphologically eclectic genera with a diverse occurrence and structure of a glandular appendage in the anther, and the shape and location of this gland in the anther. Among 50 species analyzed within the ADA clade, 21 exhibit secretory cavities, two secretory ducts, and nine phenolic idioblasts, for a total of 64% species with a secretory structure in the anther apex.
Anatomical analyses of the anther in a longitudinal section showed that in most species analyzed, the gland is a secretory cavity with lumen shapes ranging from spherical to oval (see Table 1). Myroxylon balsamum and M. peruiferum, two species of the Amburaneae subclade, are the exceptions. The gland in the anther exhibits an elongated lumen so that, the term, secretory duct, becomes more appropriate. Variations in the lumen shape and, consequently, the difficulties generated in the typification of the gland, have been extensively explored in the literature, especially in studies with the leaf [15,20] and the stem [14,15,18,19].
The glandular appendage in the anther is most evident in the species of Pterodon, Dipteryx [7,13], present study, and Cordyla, which have secretory cavities with a spherical lumen, except for D. polyphylla in which the appendix is not very prominent, and the cavities have oval lumens. Similarly, Taralea cordata, T. crassifolia, and T. nudipes exhibit non-prominent anther appendages and contain a secretory cavity with an oval lumen, which is found in the region between the two thecae. In these species, the lumen of the cavity has the same shape as described for the vegetative organs of Taralea oppositifolia [15].
It is interesting to note that the secretory cavity located at the apex of the anther is subepidermal, and the epidermis cells have phenolic compounds see [13]. In Monopteryx and some species of Alexa, the cells of the anther appendix also exhibit phenolic content, although they do not have a secretory cavity. The appendix composed of phenolic cells that the anthers exhibit must be related to the floral structure, which is non-papilionaceous in the species of Alexa and Monopteryx. In these cases, the wing petals are reduced, the keel petals are united and opened, and the free stamens are exposed. The presence of phenolic compounds in the anther apex may be associated with the defense against herbivory or UV radiation since anthers are not protected by the petals as in a papilionaceous flower [34][35][36][37].
An interesting fact is the association between a secretory cavity in the anther and the leaf. Secretory cavities are present at the anther apex of closely related species such as  [20,21] which demonstrates that these structures also occur in the floral organs of these species.

Evolutionary History of the Presence of a Secretory Cavity in the Anther of the ADA Clade
Our hypotheses were postulated to explain the occurrence of glands in anthers of the ADA clade species and have two robust explanations: (1) the appearance of the anther glands in the Amburaneae and Dipterygeae subclades or (2) their loss in some species of Amburaneae and Dipterygeae and all species of Angylocaleceae (see Figure 9).
Considering that the secretory cavities are present in other genera of the Amburaneae subclade, it is concluded that they are not a synapomorphy of the Dipterygeae subclade, as suggested by Leite et al. [7].
In the Dipterygeae subclade, our data suggest that secretory cavities may have been acquired in Dipteryx + Pterodon and some species of Taralea. A phenolic glandular appendix may have been acquired in Monopteryx, a sister group of Dipteryx, Pterodon, and Taralea (see distribution in Figure 9). The glandular appendix with a phenolic epidermis in Dipteryx, Pterodon, and Taralea could be a remnant of the Monopteryx phenolic appendage.
The Amburaneae subclade is remarkable because of its high level of floral diversity, production of coumarins (Amburana), red resin from bark and twigs (Dussia), balsams (Myrocarpus, Myrospermum, Myroxylon), and punctate glandular leaves of several genera (Cordyla, Mildbraediodendron, Myrocarpus, Myrospermum, and Myroxylon) [27,38,39]. The presence of glands at the anther apex is also noteworthy. Our data suggest that secretory cavities/ducts may have been acquired in the well-supported clades Myroxylon + Myrocarpus (non-papilionaceous flowers) and Myrospermum (papilionaceous flower). Another interesting fact is the presence of a secretory duct only in Myroxylon, and therefore, an autapomorphy of Myroxylon. Although Cordyla and Mildbraediodendron have a swartzioidlike floral morphology [12,38], the flowers have an entire calyx, no petals, and numerous free stamens, only Cordyla exhibits a secretory cavity in the anther apex. In contrast, the genus Amburana, sister to Cordyla + Mildbraediodendron, does not have a secretory cavity in the anther and has a one-petalled corolla and 10 free stamens. The absence of a secretory cavity in the anther apex of the genera Petaladenium and Dussia reflects their positioning in the phylogenetic tree as sister genera. They also present a papilionaceous corolla and basally united stamens [10]. A peculiar characteristic of Petaladeninum urceoliferum is its wing petals with glands, while in Dussia (its sister genus), the glands are found on the bract and bracteoles [9], although there are no anatomical studies on the composition of these structures.
In the Angylocalyceae subclade, our data suggest that secretory cavities were not acquired in the genera Alexa, Angylocalyx, Castanospermum, and Xanthocercis, defining them as a sister group of the Amburaneae and Dipterygeae subclades. An appendix producing phenolic compounds may have been acquired in some Alexa species, a sister group of Castanospermum, both with similar floral morphology, non-papilionaceous corolla, and 10 free stamens [12]. Angylocalyx and Xanthocercis, sister genera, exhibit a distinct floral morphology [12].
Other relevant data are the occurrence of secretory cavity/duct vs. anther appendage shape (see Figure 10) and lumen shape vs. position of cavity/duct in the anther (see Figure 11). It is likely that the anthers with a prominent apex also exhibit secretory cavities with a spherical lumen, different from those with a non-prominent apex, which exhibit secretory cavities more internalized in the anther, between the thecae, with the lumen being oval. Thus, we suppose that the shape of the anther is related to the lumen shape in species with a secretory cavity in the anther (see Figures 10 and 11). Another interesting result is the lumen shape in the clades Myrocarpus + Myrospermum, and Myroxylon. Myrocarpus+ Myrospermum exhibit secretory cavities more internalized in the anther between the thecae, with an oval lumen and a prominent/non-prominent apex, respectively, different from Myroxylon, which exhibits a secretory duct with a rounded lumen in its apical portion and is elongated in the lower portion between the thecae following the shape of the anther apex.
The secretory cavities of Dipteryx (except D. polyphylla) and Pterodon are anatomically more similar to each other than to those of Taralea cordata, T. crassifolia and T. nudipes, confirming previous data obtained about the flower [7], leaf, stem [15] and leaflets [16] and corroborating the phylogeny data (see LPGW [1]). This fact reflects the topology of a phylogenetic tree in the subclades, which places them as sister groups see [1,12,22].

Evolutionary Significance of the Corolla, Type of Androecium vs. Presence of a Secretory Cavity/Duct in the Anther
Our data plotted in phylogeny have not yet made it possible to correlate the type of corolla and androecium with a secretory cavity/duct in the anther. The presence of a secretory cavity/duct has been reported in species with apetalous, non-papilionaceous flowers with numerous free stamens, including Cordyla africana, C. haraka and C. madagascariensis. Myrocarpus emarginatus, M. fastigiatus, and M. frondosus exhibit five undifferentiated petals with 10 basally united stamens, Myrospermum frutescens exhibits a papilionaceous corolla with 10 free stamens; Myroxylon balsamum and M. peruiferum exhibit a non-papilionaceous corolla with the widely oval banner and 10 free stamens. Therefore, a correlation with pollination seems plausible.
In Dipteryx alata and Pterodon pubescens, species of the Dipterygeae subclade, the anther glands consist of a cavity secreting sticky substances (oleoresins and polysaccharides) that play a key role during the flower's lifespan by aggregating pollen grains and attaching them to the floral visitor's body, besides maximizing the pollen release mechanism that is intermediate between the valvular and the explosive [13]. The same mechanism probably occurs in Myrospermum frutescens because these species are pollinated by insects [42]; Myrocarpus frondosus has a non-papilionaceous corolla but is pollinated by insects [43], probably aggregating pollen grains.
In Myroxylon peruiferum, the ducts secrete a substance that aggregates pollen grains; however, when in contact with air, the resinous content secreted together with the pollen grains hardens, probably acting on pollination by sunbirds (personal observation). The same must occur in Cordyla africana, which has petaliferous flowers rich in nectar that are pollinated by sunbirds [44].
Species with phenolic idioblasts at the anther apex, such as those of Alexa and Monopterx, are ornithophilous [27] and entomophilous [45], respectively. There are also reports of sphingophily [46,47] and chiropterophily [48] for Alexa. The lack of information on the pollination biology for these species makes it difficult to understand the function of these glands in early-diverging papilionoids.
However, analyzing the corolla shape and the presence of a secretory structure in the anther concerning the pollinator (insect or bird), probably the corolla type associated with the material exuded by the secretory structure at the anther apex at different proportions found in each species acts by favoring the different pollinators.

Outlook
We studied the evolution of the anther's glandular appendage in early-diverging papilionoid genera, reporting this condition in a large number of species.
Samples of flowers and floral buds (1 or/and 2) were obtained from herbarium specimens (Table S1) and treated with 2% KOH solution for 2 h, washed several times in distilled water [56], and stored in 70% ethanol. The anthers were removed and prepared for observations by scanning electron microscopy (SEM) and light microscopy (LM).

Scanning Electron Microscopy
For SEM analysis, anthers were critical point dried in a Balzers CPD 030 dryer (Balzers, Liechtenstein), mounted on aluminum stubs with colloidal carbon, coated with gold in a Bal Tec SCD 050 sputter coater, and observed with a Jeol JSM 6610LV scanning electron microscope (Tokyo, Japan).

Phylogenetic Analysis
The evolution of the glands in anthers from the ADA clade was investigated based on a recent phylogenetic hypothesis [1]. The characters selected to compose the data matrix were the absence or presence of secretory cavity/ducts, anther appendage shape (prominent, not prominent), anther appendix position (apical, distal, apical/distal), lumen shape (spherical, oval, elongated), and absence or presence of phenolic compound at the apex of the anther (Table 2). We used the Mesquite program [59] to map the selected characters (Table 2) on the RAXML tree obtained by the Legume Phylogeny Working Group (LPWG) [1]. For this purpose, we chose "trace character history", selecting the option "parsimony ancestral states". Table 2. Morphological characteristics evaluated in taxa of the Amburaneae, Angylocaleceae, Dipterygeae subclades, Ateleia, Candolleodendron, Cyathostegia, Swartizia and Uleanthus. Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable to this article.